Absorption of Free Radicals of New S-Derivatives (1,2,4-Triazole-3(2H)-yl)methyl)thiopyrimidines ^†

Abstract

At the current stage of organic chemistry development, various fundamental synthetic approaches have been developed for the synthesis of 1,2,4-triazole and pyrimidine scaffolds, which demonstrate diverse biological effects. The relevance of this research lies in the combination of two pharmacophore fragments in one molecule—a pyrimidine and an azole heterocycle—connected by a thiomethylene bridge, which is expected to improve solubility and enhance known biological properties, as well as introduce new ones. This study presents the synthesis of compounds and investigates their antiradical activity applying the DPPH free radical method. Three compounds demonstrate greater activity than the reference drug, the natural antioxidant ascorbic acid.

1. Introduction

Antioxidants are substances that prevent and mitigate damage caused by free radicals by transferring electrons from the antioxidants to these reactive species [1]. They also convert free radicals into waste products that are subsequently eliminated from the body. Therefore, evaluating such properties remains an interesting and valuable task, particularly in the search for promising synthetic antioxidants derived from azole heterocycles.

Pyrimidine is an aromatic heterocyclic compound containing nitrogen atoms at the 1st and 3rd positions, and plays a crucial role in forming the backbone of various biologically active compounds. It is a structural unit of DNA and RNA and is essential in various biological processes. Common pyrimidines include uracil, cytosine, and thymine. Pyrimidines exhibit a spectrum of biological actions [2], including antiviral, antitumor, antimicrobial [3], anti-inflammatory, analgesic [4], antioxidant [5], and antimalarial properties.

Pyrimidine is used as a starting material for synthesizing a wide range of heterocyclic compounds and for producing new molecules. Pyrimidine ring complexes, when combined with various heterocyclic fragments, have been shown to be vital ingredients in pharmaceutical and veterinary products.

The 1,2,4-triazole nucleus is a highly promising azole heterocycle, and compounds derived from its chemical transformations have various biological, pharmaceutical, and clinical applications [6]. It is widely accepted that modifying the structure of azole heterocycles can boost their effectiveness and lower toxicity.

An increase in solubility, enhancement of known biological properties, and the emergence of new biological activities can be achieved by combining two pharmacophoric fragments in one molecule—a pyrimidine and an azole heterocycle—connected by a thiomethylene bridge. Therefore, we selected new S-derivatives of (1,2,4-triazol-3(2H)-yl)methyl)thiopyrimidines for research. Derivatives of (1,2,4-triazol-3(2H)-yl)methyl)thiopyrimidines are known to exhibit anticonvulsant effects and to treat nervous system disorders [7].

2. Materials and Methods

Chemistry. Using a Bruker AC-400 spectrometer (400 MHz), the ¹H NMR spectra were measured with DMSO-d₆ serving as the solvent and TMS as the internal reference (Agilent Technologies, Santa Clara, CA, USA). The LC-MS analysis was performed on a high-performance Agilent 1260 Infinity HPLC System, with a diode array detector and proton ionization for detection. Elemental analysis (C, H, N, S) was performed using the ELEMENTAR vario EL cube, with sulfanilamide as the standard. The capillary technique was applied to determine the melting points, using the Stanford Research Systems Melting Point Apparatus 100 (SRS, Sand-Hills, SC, USA). The reagents employed in this research were obtained from Sigma-Aldrich (Merck), Steinheim, Germany.

Following a previously reported procedure [8], the compounds were synthesized, with their physical constants aligning with documented values.

Obtaining of 4-methyl-5-((pyrimidin-2-ylthio)methyl)-4H-1,2,4-triazole-3-thiol 1 (general methods). A mixture of 10 mmol 2-(pyrimidin-2-ylthio)acetohydrazide, 10 mmol sodium hydroxide, and 50 mL purified water was boiled for 2 h. When the mixture reached room temperature, 2 mL of concentrated acetic acid was added to the filtrate. The formed precipitate was then subjected to filtration and washed with distilled water. For further analysis, the product was purified via recrystallization from DMF, yielding a light-yellow powder. The compound is soluble in aqueous alkali solutions, DMF, and 1,4-dioxane.

4-Methyl-5-((pyrimidin-2-ylthio)methyl)-4H-1,2,4-triazole-3-thiol (1). Yield 72%, light-yellow powder, mp 266 °C (DMF). ¹H NMR, δ, ppm. (J, Hz): 3.55 (s, 3H, -N-CH₃), 4.43 (s, 2H, -CH₂-), 7.19 (t, J = 4.4 Hz, 1H, Ar), 8.53 (d, J = 4.4 Hz, 2H, Ar), 12.83 (s, 1H, -SH). Mass spectrum, m/z (I_rel, %) 240 [M + H]⁺ (100). Anal. calcd. for C₈H₉N₅S₂: C: 40.15%; H: 3.79%; N: 29.26%; S: 26.79%; found: C: 40.11%; H: 3.82%; N: 29.35%; S: 26.71%.

Obtaining of S-alkyl derivatives 4-methyl-5-((pyrimidin-2-ylthio)methyl)-4H-1,2,4-triazole-3-thiols 2–4 (general methods). A solution of 5 mmol 4-methyl-5-((pyrimidin-2-ylthio)methyl)-4H-1,2,4-triazole-3-thiol and 5 mmol sodium hydroxide dissolved in 15 mL of i-propanol is prepared. To this, 5 mmol of the halogen derivative is added. The mixture is heated for 2 h, then cooled, and the sediment is filtered and washed with purified water. The product is crystallized from methanol for analysis. The crystalline substances (2-4) are yellow or brown, insoluble in water, and soluble in organic solvents.

2-((4-Methyl-5-((pyrimidin-2-ylthio)methyl)-4H-1,2,4-triazol-3-yl)thio)acetic acid (2). Yield 1.16 g (78%), white powder, mp 184 °C (MeOH). ¹H NMR, δ, ppm. (J, Hz): 3.59 (s, 3H, -N-CH₃), 4.08 (s, 2H, -CH₂-COO), 4.54 (s, 2H, -CH₂-), 7.20 (t, J = 3.7 Hz, 1H, Ar), 8.52 (d, J = 3.7 Hz, 2H, Ar), 11.36 (s, 1H, -COOH). Mass spectrum, m/z (Irel, %) 298 [M + H]⁺ (100). Anal. calcd. for C₁₀H₁₁N₅O₂S₂: C: 40.39%; H: 3.73%; N: 23.55%; S: 21.56%. Found: C: 40.32%; H: 3.76%; N: 23.58%; S: 21.52%.

Methyl 2-((4-methyl-5-((pyrimidin-2-ylthio)methyl)-4H-1,2,4-triazol-3-yl)thio)acetate (3). Yield 1.13 g (73%), white powder, mp 169 °C (MeOH). ¹H NMR, δ, ppm. (J, Hz): 3.72 (s, 3H, -N-CH₃), 4.07 (s, 2H, -CH₂-COO), 4.54 (s, 2H, -CH₂-), 7.20 (t, J = 3.7 Hz, 1H, Ar), 8.52 (d, J = 3.7 Hz, 2H, Ar). Mass spectrum, m/z (I_rel, %) 312 [M + H]⁺ (100). Anal. calcd. for C₁₁H₁₃N₅O₂S₂: C: 42.43%; H: 4.21%; N: 22.49%; S: 20.59%. Found: C: 42.18%; H: 4.20%; N: 22.54%; S: 20.67%.

2-((4-Methyl-5-((pyrimidin-2-ylthio)methyl)-4H-1,2,4-triazol-3-yl)thio)acetamide (4). Yield 1.11 g (75%), white powder, mp 197 °C (MeOH). ¹H NMR, δ, ppm. (J, Hz): 3.59 (s, 3H, -N-CH₃), 4.01 (s, 2H, -CH₂-COO), 4.54 (s, 2H, -CH₂-), 7.05 (s, 2H, -NH₂), 7.20 (t, J = 3.7 Hz, 1H, Ar), 8.52 (d, J = 3.7 Hz, 2H, Ar). Mass spectrum, m/z (I_rel, %) 297 [M + H]⁺ (100). Anal. calcd. for C₁₀H₁₂N₆OS₂: C: 40.53%; H: 4.08%; N: 28.36%; S: 21.64%. Found: C: 40.38%; H: 4.16%; N: 28.96%; S: 21.50%.

Antiradical activity. Free radical absorption was measured using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical test [9]. To prepare the solution, an exact weight of the substance (0.001 M) was transferred into a 25.00 mL volumetric flask, dissolved in DMSO, brought to the mark, and then mixed thoroughly. Following this, 1.00 mL of the solution was introduced into a 10.00 mL volumetric flask (0.0001 M), diluted to the mark with DMSO, and well mixed. After that, 2.00 mL of the resulting solution was placed in a test tube, combined with 2.00 mL of a 0.1 mM DPPH solution in methanol (Sigma-Aldrich, Steinheim, Germany), and the test tube was sealed tightly. The tubes were shaken thoroughly and left in the dark for 30 min at room temperature. Absorbance was measured at a wavelength of 516 nm. The control was a solution of 2.00 mL of 0.1 mM DPPH in the presence of 2.00 mL of methanol, and the standard was ascorbic acid. The activity of scavenging free radicals was represented as a percentage of inhibition and determined by Formula (1):

$$\%\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}={\displaystyle \frac{\left({\mathrm{A}}_0-{\mathrm{A}}_1\right)}{{\mathrm{A}}_0}}·100,$$

(1)

where A₀ represents the absorption coefficient of the control sample, and A₁ denotes the absorption coefficient of the test sample. The absorption of the studied solutions was measured in aqueous organic solutions, and the absorption maximum at 516 nm was recorded using a SPECORD 250 spectrophotometer.

3. Results

A well-established method for synthesizing 5-substituted-1,2,4-triazole-3(2H)-thiones involves the formation of intermediate carbothioamides, followed by heterocyclization in an alkaline medium [9]. The precursor, pyrimidine-2-thione, was prepared through a [3 + 3] cyclization of thiourea with 1,1,3,3-tetraethoxypropane. Subsequently, ethyl 2-(pyrimidin-2-ylthio)acetate was synthesized via an alkylation reaction in acetone, using K₂CO₃ as the base (Figure 1).

The ester was subjected to hydrazinolysis, yielding a hydrazide, which subsequently reacted with methyl isothiocyanate in ethanol to form the intermediate carbothioamide. Cyclization was achieved by stirring the mixture with an aqueous NaOH solution for 2 hours on a magnetic stirrer. The solution was treated with glacial acetic acid, resulting in the formation of 5-((pyrimidin-2-ylthio)methyl)-4-methyl-4H-1,2,4-triazole-3-thiol (1). It is well-established that substituents on the sulfur atom in 1,2,4-triazole-3(2H)-thiones significantly enhance their biological activity. Subsequently, the S-derivatives of (1,2,4-triazol-3(2H)-yl)methylthiopyrimidines were synthesized. Alkyl and acyl derivatives (2–4) were obtained by reacting the parent thione (1) with the corresponding halogen derivative in a polar solvent—ethanol—with the incorporation of an equal molar quantity of NaOH.

Among the various groups of antioxidants that function through distinct pathways or employ varied mechanisms of action, the most important role is played by antiradical antioxidants—substances that interact with free radicals to form products incapable of continuing oxidation chain reactions or that reduce the reaction rate. The antiradical activity of the prepared compounds was assessed through a free radical scavenging assay employing 1,1-diphenyl-2-picrylhydrazyl (DPPH) (

Table 1. Antiradical activity and absorption coefficients of 1,2,4-triazole derivatives.

Compounds	Absorption Coefficient, A	% Antiradical Activity
Control	0.4305	–
Ascorbic acid	0.2959	31.26
1	0.1864	56.7
2	0.0681	83.86
3	0.4304	−1.99
4	0.2572	39.05

). The results indicated that the compounds exhibited significant antiradical activity.

The absorption spectra of 1,1-diphenyl-2-picrylhydrazyl (DPPH) with the compounds are shown in Figure 2, with absorption values at 516 nm. The negative value of antiradical activity (%) may be attributed to two factors: the similarity between the absorption spectra of the substance and DPPH, causing overlapping absorption bands, or an increase in the degradation of the compound, resulting in the release of free radicals. This is evident in the case of a compound with an ethanoic acid ester group at the 5-position of 1,2,4-triazol-3(2H)-thione, and may be due to a non-ideal electronic configuration of the framework for reducing free radicals via dissociation.

It is important to note that three compounds (1, 2, 4) demonstrate higher activity than the reference preparation, the natural antioxidant ascorbic acid. This high activity may be related to the existence of pharmacophore fragments, specifically the pyrimidine skeleton and the sulfur atom linked to 1,2,4-triazole. A more detailed analysis of the compounds suggests a relationship between "antiradical activity and structure," with the enhanced activity attributed to the presence of free proton donors in the compounds, particularly in the protonated atoms of the pyrimidine ring and in proton donors within the carboxylic acid and amide residues.

4. Conclusions

The negative value of antiradical activity (%) can be attributed to two factors: the similarity between the absorption spectra of the substance itself and DPPH, causing overlap of the absorption bands, or the increased degradation of the compound, leading to the release of free radicals. Notably, three compounds (1, 2, 4) exhibit higher activity than the reference drug, the natural antioxidant ascorbic acid. This high activity may be related to the existence of pharmacophore fragments, specifically the pyrimidine skeleton and the sulfur atom linked to 1,2,4-triazole.